Hepatotropic conjugates of antiviral drugs carriers thereof and pharmaceutical compositions containing them

ABSTRACT

The present invention refers to conjugated compounds of antiviral drugs having hepatotropic activity, methods of making these compounds, and compositions thereof.

This is a continuation of application Ser. No. 08/374,726, filed Mar.17, 1995, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention refers to compounds with antiviral activity and,more particularly to conjugated compounds of antiviral drug withcarriers having hepatotropic activity.

2. Description of the Related Art

In the treatment of the infection caused by viruses, the side effectsproduced by antiviral drugs can be reduced with the adoption of thechemotherapeutic lysosomotropic approach (Balboni P G, Minia A, Grossi MP, Barbanti-Brodano G, Mattioli A, Fiume L., Activity of albuminconjugates of 5-fluorodeoxyuridine and cytosine arabinoside onpoxviruses as a lysosomotropic approach to antiviral chemotherapy,Nature 1976; 264: 181-183).

This consists in conjugating the drug to a macromolecule that isselectively captured from the infected cells and transported in thelysosomes therefrom.

If, as desired, the lysosomal enzymes break the linkage between thecarrier and the drug, the latter results as being concentrated in apharmacological active form within the infected cells.

    AZTMP=3-azido-3-deoxythymidine

    ACV=acyclovir

The chronic hepatitis by B (HBV) virus and by C (HCV) virus are a propertarget for this chemotherapeutic approach because: (a) these virusesgrow especially in the hepatocytes; (b) hepatocytes specifically bringinside and transport in the lysosomes some glycoproteins with galactoseresidues that can therefore function as hepatotropic vectors of drugs;(c) the conjugates of drug/glycoproteins can easily come into contactwith the surface of the hepatocites inasmuch the hepatic sinusoid arenot a barrier for proteins.

Following this approach and to reduce its neurotoxic side effects, thearabinoside adenine monophosphate antiviral drug (ara-AMP), activeagainst HBV (Jacyna M R, Thomas H C., Antiviral therapy: Hepatitis B.,Brit Med Bull 1990; 46: 369-382), has been conjugated with asialophetuin(AF) (Fiume L; Mattioli A, Busi C, Balboni P G, Barbanti-Brodano G, DeVries J, Altman R, Wieland Th., Selective inhibition of Ectromelia virusDNA synthesis in hepatocytes by adenine-9-β-D-arabinofuranoside (ara-A)and adenine-β-D-arabinofuranoside 5'-monophosphate (ara-AMP) conjugatedto asialofetuin, FEBS LEtters 1980; 116: 185-188) and with thelactosaminated albumin (L-SA) (Fiume L, Busi C, Mattioli A, Balboni P G,Barbanti-Brodano G., Hepatocyte targeting ofadenine-9-β-D-arabinofuranoside 5'-monophosphate (ara-AMP) coupled tolactosaminated albumin, FEBS Lett 1981; 129: 261-264; Fiume L, Bassi B,Busi C, Mattioli A, Spinosa G., Drug targeting in antiviralchemotherapy. A chemically stable conjugate of9-β-D-arabinofuranosyladenine 5'-monophosphate with lactosaminatedalbumin accomplishes a selective delivery of the drug to liver cells,Biochem Pharmacol 1986; 35: 967-972). In mice, both these two carriershave brought about a hepatic targeting of the drug.

The L-SA has a great advantage on AF: infact, the conjugates preparedwith homologous lactosaminated albumin (that is, of the same species),when introduced intravenously, do not induce the antibodies formation(Fiume L, Mattioli A, Busi C, Spinosa G, Wieland Th., Conjugates ofadenine-9-β-D-arabinofuranoside monophoshate (ara-AMP) withlactosaminated homologous albumin are not immunogenic in the mouse,Experientia 1982; 38: 1087-1089; Fiume L., Busi C., Preti P., Spinosa G.Coniugates of ara-AMP with lactosaminated albumin: a study on theirimmunogeneticity in mouse and rat. Cancer Drug Delivery 1987; 4:145-150). In woodchuck with hepatitis by WHV (Ponzetto A, Fiume L,Forzani B, Song S Y, Busi C, Mattioli A, Spinelli C, Marinelli M,Smedile A, Chiaberge E, Bonino F, Gervasi G B, Rapicetta M, Verme G.,Adenine arabinoside monophosphate and acyclovir monophosphate coupled tolactosaminated albumin reduce woodchuck hepatitis virus viremia at doseslower than do the unconjugated drugs, Hepatology 1991; 14: 16-24) and inpatients with chronic infection by HBV, (Fiume L., Torrani Cerenzia M R,Bonino F, Busi C, Mattioli A, Brunetto M R, Chiaberge E Verme G.Inhibition of hepatitis B virus replication by vidarabine monophosphateconjugated with lactosaminated serum albumin, Lancet 1988; 2: 13-15.Torrani Cerenzia M R, Fiume L, Busi C, Mattioli A, Di Stefano G, GervasiG B, Brunetto M R, Piantino P. Verme G, Bonino F. Inhibition ofhepatitis B virus replication by adenine arabinoside monophosphatecoupled to lactosaminated albumin. Efficacy, minimal effective dose andplasma clearance of conjugate. J Hepatol--1994; 20: 307-309), theara-AMP conjugated to the L-SA has inhibited the viral replication atdoses 3-6 times lower than those of the free drug.

The conjugate with L-SA should be administered intravenously owing tothe high volume needed for the injection and because, by other waysantibodies are produced, and this results in a not good patientcompliance in long-lasting treatments.

A hepatotropic carrier of ara-AMP and of other antiviral drugs allowingthe intramuscular administration of the corresponding conjugates wouldtherefore be a remarkable improvement from the therapeutic point ofview.

It is also known that a basic polyaminoacid, the poly-L-lysine, with onethird of the amino-groups substituted with galactose residues, ifintravenously administered, performs a hepatic targeting of ara-AMP(Fiume L, Bassi B, Busi C, Mattioli A, Spinosa G, Faulstich H.,Galactosylated poly(L-lysine) as a hepatotropic carrier of9-β-D-arabinofuranosyladenine 5'-monophosphate, FEBS Letters 1986; 203:203-206). Moreover, the poly-L-lysine, when all or large part of itsε-amino groups are substituted, does not form antibodies even whenadministered by ways different from the intravenous injection (Levine BB, Studies on antigenicity. The effect of succinylation of ε-aminogroups on antigenicity of benzylpenicilloyl-poli-L-lysine conjugates inrandom-bred and in strain 2 Guinea pig, Proc Soc Exptl Biol Med 1964;116: 1127-1131; Sela M. Immunological studies with syntheticpolypeptides, Advan Immunol, 1966; 5: 29-129).

It has now been found that if in the poly-L-lysine most of the ε-aminogroups are substituted with the galactose and with one of the antiviraldrugs known for performing their activity against hepatic viruses, threevery important therapeutic effects are produced in mice:

(i) the conjugate loses the poly-L-lysine high toxicity whichdistinguished the previous galactosylated poly-L-lysine-ara-AMPconjugates (Fiume et al, FEBS Letters 1986, 203, 203-206) in which mostthe ε-amino groups remained unsubstituted;

(ii) the antiviral drug hepatic targeting is brought on even if theconjugate is not administered intravenously, and particularly, byintramuscular injection.

(iii) the repeated conjugate administration by intramuscular andintravenous injection does not produce antibodies.

The therapeutic importance of this property will be evident if it isconsidered that the possibility to achieve the hepatic targeting byintramuscular injection not only involves to utilize its interestingpeculiar characteristics already previously mentioned in relation to thelysosomotropic approach (i.e. drastic reduction of the side effectsrelevant to the toxicity of the antiviral drugs), but also as much asimportant to make much more comfortable for the patient to undergo thechemotherapeutic-treatment which, as a rule in the viral chronichepatitis, is long lasting.

In the preferred definition, the basic poliaminoacid is selected betweenpoly-L-lysine and poly-L-ornithine and the drugs among those known fortheir activity against the hepatic viruses, and particularly amongara-AMP, acyclovir, ribavirin, azidothymidine, and the like.

The present invention refers therefore to the use, as carrier ofantiviral drugs, of a basic, galactosylated polyaminoacid, saidpolyaminoacid being characterized in that most of the amino groups aresubstituted with molecules of the drug and with galactose molecules.

This high degree of substitution eliminates the acute toxicity of boththe basic polyaminoacids and the conjugates of poly-L-lysine previouslypublished (Fiume et al. FEBS Letters 1986; 203: 203-206) in which lessthan 50% of ε-amino groups were substituted by the drug and by thegalactosyl residues.

The preparation of the conjugated compounds according to the presentinvention provides a two-steps procedure:

(a) conjugation of the basic polyaminoacid with antiviral drug or withgalactose residues and

(b) subsequent conjugation of the conjugate resulting from step (a) withgalactose residues or antiviral drugs residues, respectively. In theprevious general definition it can be noticed that the two conjugationsteps may be inverted.

As a rule, the choice is suggested by the polyaminoacid molecular weightin a sense that, with lower molecular weights, it is preferred to carryout first the conjugation with the drug and the conjugation withgalactose thereafter.

BRIEF DESCRIPTION OF THE DRAWINGS

The following determinations are referred to the enclosed figures inwhich:

FIG. 1 shows the chromatographic diagram on Bio Gel P 10 of theLac-poly-L-lysine-ara-AMP (compound 4 of table 1).

FIGS. 2A-2H show the radioactivity distribution in liver (▪), in spleen(), in intestine (◯) and in brain (□) of female Swiss mice (28-30 g) towhich the compounds of the invention (Table 1) and the unconjugatedcompounds for comparison have been administered by intramuscularinjection.

FIG. 3 shows the chromatographic diagram on Bio Gel P 2 of liverextracts of mice injected with the compound 14 of Table 1.

FIG. 4 shows the organ radioactivity distribution in mice afterintramuscular injection of LAT- ³ H!poly-L-ornithine-ara-AMP.

FIG. 5 shows the gel permeation chromatography of compound 9.

FIGS. 6A-6E show the organ distribution of compounds having highmolecular weight conjugates.

DESCRIPTION OF PREFERRED EMBODIMENTS

In the preferred embodiment of the process according to the presentinvention the drug conjugation is carried out in a way known by itselfthrough the imidazolate of the antiviral drug in its monophosphate formand by performing the conjugation at alkaline pH (Fiume L, Busi C, DiStefano G, Mattioli A, Coupling of antiviral nucleoside analogs tolactosaminated human albumin by using the imidazolides of theirphosphoric esters--Analyt Biochem 1993, 212: 407-411).

The galactose residues are preferably conjugated (step b) by reductivelactosamination in the presence of sodium cyanoborohydride (Schwartz BA, Gray G R Proteins containing reductively aminated disaccharidesSynthesis and chemical characterization--Arch Biochem Biophys 1977; 181:542-549).

As a non limiting example, the examples of preparation related to theuse of poly-L-lysine and of poly-L-ornithine as a carrier, and ofara-AMP, acyclovir ribavirin and azidothymidine as antiviral drugs arereported, being understood that similar procedures are followed forpreparing conjugates with other known antiviral drugs.

Conjugates with both low and high molecular weight basic polyaminoacidshave been prepared.

I. LOW MOLECULAR WEIGHT CONJUGATES A. Conjugates with poly-L-lysine A.1.Preparation

In a commercial composition (Sigma) of poly-L-lysine with a molecularweight of 1000-4000 Da and with an average polymerization grade of 14,the polymers with a molecular weight lower than 1800 were removed bymeans of gel filtration on a P2 Bio Gel column eluted with 0, 2 M NH₄HCO₃. The polymers excluded from the column, after lyophilization, havebeen utilized to prepare the compounds shown in the following table 1.

In all the conjugates the galactose residues have been linked to theε-amino groups by reductive lactosamination in the presence of sodiumcyanoborohydride (Schwartz B A, Gray G R., Arch. Biochem Biophys 1977;181: 542-549).

Compound 1

The poly-L-lysine has been labeled with ³ H!formaldehyde following theJentoft and Dearborn method (Jentoft N, Dearborn D G., Protein labellingby reductive alkylation, Methods Enzymol 1983; 91: 570-579). Thereaction mixture contained 44 μCi of ³ H!formaldehyde/ml. The ³H!poly-L-lysine has been isolated by means of gel filtration on a P2 BioGel column and subsequent lyophilization.

                                      TABLE 1    __________________________________________________________________________    CHARACTERISTICS OF LOW MOLECULAR WEIGHT POLY-L-LYSINE CONJUGATES.                                   % ε-NH.sub.2 groups                                            Specific                     μg lactose                            μg drug                                   substituted by                                            Activity    Compounds        mg compound                            mg compound                                   Lactose                                        Drug                                            (dpm × 10.sup.3 /mg)    __________________________________________________________________________    1)  .sup.3 H!poly-L-lysine                     0      0      0    0   1200    2)  .sup.14 C!LAC-poly-L-lysine                     721    0      92   0   860    3)  .sup.14 C!LAC-poly-L-lysine                     540    206    72   28  500    4) LAC-poly-L-lysine-ara-AMP                     493    240    66   33  0    5) LAC- .sup.3 H!poly-L-lysine-ara-AMP                     573    147    72   20  36000    6) LAC-poly-L-lysine-ara- .sup.3 H!AMP                     613    176    73   22  7500    7) LAC-poly--L-lysine- .sup.3 H!ACVMP                     638    81     80   12  440    __________________________________________________________________________

Compound 2

The galactose has been linked to polylysine by reductive lactosaminationin the presence of sodium cyanoborohydride. 20 mg of poly-L-lysine weredissolved in 2 ml of a 0.1 M boric acid/borax buffer (pH 8.5). 80 mg ofalpha-lactose, conteining 50 μCi of D-glucose-1-¹⁴ C! lactose(Amersham), and 50 mg of NaBH₃ CN have been added. The mixture has beenincubated at 37° C. for 48 hours and the ¹⁴ C!Lac-poly-L-lysine isolatedas for the compound 1. The lactose contents was measured with the Duboiset al. method (Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F.,Colorimetric method for determination of sugar and related substances,Anal Chem 1956; 28: 350-356) and referred to the dry weight of thecompound.

Compounds 3 and 4

The ara-AMP has been conjugated by means of its imidazolate (Fiume L,Busi C, Di Stefano G, Mattioli A., Analyt Biochem--1993; 212:407-411).

This procedure is more effective than that employing the water solublecarbodiimides and avoids the chemical side reactions produced by thesesubstances.

The ara-AMP imidazolate has been prepared with the Lohrmann and Orgelmethod (Lohrmann R, Orgel L E, Preferential formation of (2'-5')-linkedinternucleotide bonds in non-enzymatic reactions, Tetrahedron 1978; 34:853-855). Poly-L-lysine was dissolved (50 mg/ml) in a 0.1 M NaHCO₃ /Na₂CO₃ buffer, pH 9.5. After the addition of the ara-AMP imidazolate (75mg/ml), the pH was readjusted at 9.5 with HCl and the mixture incubatedfor 48 hours at 37° C. The conjugates have been isolated as the compound1; the ara-AMP content was determined spectrophotometrically andreferred to the dry weight of the conjugates, which were furtherlactosaminated (for compound 4 using unlabelled lactose).

Compound 5

The poly-L-lysine/ara-AMP conjugate, obtained as indicated for compounds3 and 4, has been labelled with ³ H!formaldehyde (100 mCi/mmole).

The reaction mixture contained 2800 μCi ³ H!formaldehyde/ml. The labeledconjugate has been then lactosaminated as described above. Thisconjugate was used as antigen in determining the antibodies with theMinden and Farr method (Minden P, Farr R S, The ammonium sulphate methodto measure antigen-binding capacity, Weir D M ed., Handbook ofExperimental Immunology, Blackwell, Oxford 1973; 15.1-15.21;).

Compound 6

The conjugation of tritiated ara-AMP in this compound was performedusing 1-ethyl-3-(dimethyl aminopropyl)carbodiimide (ECDI), since theara- ³ H!AMP conversion in its imidazolate causes an almost completeloss of tritium. The poly-L-lysine was first lactosaminated as describedabove (compound 2), but reducing the reaction period from 48 to 24 hoursin order to substitute with the sugar only 2/3 of the ε-amino groups.

Afterwards, the ara- ³ H!AMP was conjugated according to the aboveprocedure (Fiume L, Bassi B, Busi C, Mattioli A, Spinosa G, FaultstichH, FEBS Letters 1986; 203: 203-206). In the preparation of thisconjugate the first step was the lactosamination to reduce the number ofthe free ε-amino groups at the time of use of the ECDI and, thereforethe possibility of polymerizing the poly-L-lysine molecules by means ofthis compound.

Compound 7

The ³ H!ACVMP employed to produce this conjugate was obtained byphosphorylation (Yoshikawa M, Kato T, Takenishi T., A novel method forphosphorylation of nucleosides to 5'-nucleotides, Tetrahedron Lett 1967;50: 5065-5068) of ³ H!ACV(triziate at the position 2 of the side chain)(NEN). The conjugate was prepared as for compounds 3 and 4, with thedifference that the incubation of the poly-L-lysine with imidazolated ³H!ACVMP lasted only 6 hours. The ³ H!ACVMP imidazolate was made with theabove cited Lohrman and Orgel method.

The conjugated product of this invention obtained in the previousexamples underwent chemical-physical determinations and biological testsaimed at finding the experimental confirmation of the therapeuticalproperties of the compounds themselves.

The following determinations are referred to the enclosed figures inwhich:

FIG. 1 shows the chromatographic diagram on Bio Gel P 10 of theLac-poly-L-lysine-ara-AMP (compound 4 of table 1).

FIGS. 2A-2H show the radioactivity distribution in liver (▪), in spleen(), in intestine (◯) and in brain (□) of female Swiss mice (28-30 g) towhich the compounds of the invention (Table 1) and the unconjugatedcompounds for comparison have been administered by intramuscularinjection.

FIG. 3 shows the chromatographic diagram on Bio Gel P 2 of liverextracts of mice injected with the compound 4 of table 1.

FIG. 4 shows the organ radioactivity distribution in mice afterintramuscular injection of LAC- ³ H!poly-L-ornithine-ara-AMP.

FIG. 5 shows the gel permeation chromatography of compound 9.

FIG. 6 shows the organ distribution of compounds having high molecularweight conjugates.

A.2. Experimental Observations

a) Average Molecular Weight of Compound 4

FIG. 1 shows the chromatography of the Lac-poly-L-lysine-ara-AMP(compound 4 of Table 1) on a Bio Gel P 10 (1.6×92) column calibratedwith dextran blue 2000 (void volume), ribonuclease (Mr 13,700) andaprotinin (Mr 6.500).

25 mg of conjugate were loaded on a 1.6×92 cm column equilibrated andeluted 0.2 M with NH₄ HCO₃. The fractions were of 2 ml. The elutionvolumes both of the conjugate and the markers (the latter indicated byarrows) were determined.

With the Whitaker method (Whitaker J R, Determination of molecularweights of proteins by gel filtratin on sephadex, Analyt Chem 1963; 35:1950-1953) it was calculated that the conjugate average molecular weightis 9100 corresponding to a carrier with 19 lysine residues and anaverage molecular weight of 2400.

b) Distribution of the Compounds in the Organs

As already mentioned, FIGS. 2A-2G show the radioactivity distribution inthe mice organs after the intramuscular injection of: A, ³H!poly-L-lysine (24 μg/g); B, ¹⁴ C!Lac-poly-lysine (24 μg/g); C, ara- ³H!AMP (5 μg/g); D, ¹⁴ C!Lac-poly-L-lysine-ara-AMP (24 μg/g); E,Lac-poly-L-lysine-ara- ³ H!AMP (28 μg/g corresponding to 5 μg/g of ara-³ H!AMP; F, ³ H!ACVMP (4 μg/g); G, Lac-poly-L-lysine- ³ H!ACVMP (50 μg/gcorresponding to 4 μg/g of ³ H!ACVMP). All the compounds were injectedin a volume of 10 μl/animal in the leg's posterior muscles using a 25 μlHamilton microsyringe.

The radioactivity contribution afforded by the plasma contained in theorgans has been calculated (Fiume L, Busi C, Mattioli A. Lactosaminatedhuman serum albumin as hepatotropic drug carrier-Rate of uptake by mouseliver, FEBS Letters 1982; 146: 42-46) and substracted.

Each datum represents the average of results achieved on 2-3 animals.

The standard error varied from 0.1 to 2% of the average values.

As illustrated after administration of ³ H!poly-L-lysine (2A), ara- ³H!AMP (2C) and ³ H!ACVMP (2F) the radioactivity quantities in liver,spleen, intestine and brain are practically equivalent. And instead,after injection of ¹⁴ C!Lac-poly-L-lysine (2B) and of conjugates of theLac-poly-L-lysine with ara-AMP and ACVMP, labeled in the lactose (2D) orin the drugs (2E, 2G), the radioactivity levels in liver are higher thanthose of the other organs.

The percentages of radioactivity measured in 1 gr of liver after ara- ³H!AMP and ³ H!ACVMP (2C,2F) administration are similar to thosecalculated after injection of an equal dose of these drugs conjugated tothe Lac-poly-L-lysine (2E,2G).

In kidneys, the radioactivity levels reach the same values as in liverone hour after the injection of the labeled conjugates (compound 3 inTable 1) or 1.5-2 times higher (compounds 6 and 7) In the subsequenttimes, the radioactivity levels of kidneys are equal or lower ascompared with those of liver. The high radioactivity levels in kidneysmight be explained by the remark that various polypeptides, afterglomerular filtration, are endocytized by the cells of kidneys proximaltubules (Maack Th, Johnson V, Kau S T, Figueiredo J, Sigulem D., Renalfiltration, transport and metabolism of low molecular weight proteins,Kidney Int 1979; 16: 251-270). The penetration into renal cells shouldnot have any effect on the chemotherapeutic index of theLac-poly-L-lysine/antiviral drugs conjugates. In fact, excludingacyclovir that tends to precipitate in the kidneys tubules (Balfour HH., Acyclovir and other chemotherapy for herpes group viral infections,Ann Rev Med 1984; 35: 279-291), the antiviral nucleosides are notparticularly toxic for the cell of this organ.

c) Digestion of Compound 3 in Liver

The rupture of the linkage between ara-AMP and the ε-amino groups of thegalactosylated poly-L-lysine within hepatic cells was demonstrated byprevious researches (Fiume L, Bassi B, Bongini A., Conjugates of9-β-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) withlactosaminated albumin: characterization of the drug-carrier bonds,Pharm Acta Helv 1988; 63 137-139). FIG. 3 shows the chromatographicprofiles on Bio Gel P 2 of the mice liver extracts, 2 and 6 hours afterthe intramuscular injection of ¹⁴ C!Lac-poly-L-lysine-ara-AMP (24 μg/g).

Female Swiss mice of 28-30 gr received by intramuscular injection theconjugate (24 μg/g in a total volume of 10 μl).

After 3 (□) or 6 () hours the mice were sacrificed (two animals foreach time) and the livers have been homogenized with 4 volumes of coldwater; 5 volumes of perchloric acid were immediately added and aftercentrifigation the supernatants were neutralized with KOH.

After 2 hours in the cold the potassium perchlorate was centrifuged andthe supernatants freeze-dried.

The freeze-dried material has been redissolved with 2 ml of H₂ O and,after centrifugation to clarify the solution, 1 ml was chromatographedon a Bio Gel P 2 (1.6×92 cm) column equilibrated and eluted with 0.2 MNH₄ HCO₃.

The radioactivity present in the fractions, wherein molecules includedin the gel and having dimensions larger than those of lactose have beeneluted, demonstrates that the poly-L-lysine is fragmented in liver eventhough its ε-amino groups are still linked to the sugar.

The symbol ◯ in the figure relates to the chromatographic profile of theextract of a homogenized liver obtained from two untreated mice and towhich the conjugate (14 μg/ml) was added just before the precipitationwith perchloric acid.

d) Production of Antibodies in Mice Treated with Compound A

Twelve female Swiss mice (weight of 28-30 g at the start of test)received the conjugate No. 4 (Table 1) administered in the posteriorleg's muscles for five days a week for four weeks in succession (dailysingle dose=700 μg/animal in 10 μl 0.9% NaCl).

The mice were bleeded form the retroorbital plexus with etherealanaesthesia a week after the last injection.

The antibodies were measured in 50 μl of serum and in triplicate bymeans of the precipitation method with ammonium sulphate according tothe Minden and Farr procedure.

As antigen the conjugate 5 (table 1) was used.

In the presence of 50 μl of serum of 5 untreated mice, the precipitateddpm have been 78±11 (SE.) In the presence of 10 μl of a mice serum ableto link either the ara-A(938 pmoles of ara-A linked by 1 ml of serum) orthe ara-AMP conjugated to the L-HSA (Fiume L, Bassi B, Busi C, MattioliA, Wieland Th., A study on the pharmacokinetics in mouse ofadenine-9-β-D-arabinofuranoside 5-monophosphate conjugated withlactosaminated albumin, Experientia 1985; 41, 1326-1328), theprecipitated dpm have been 1599±21.

In the presence of the serums of the twelve mice treated with theLac-poly-L-lysine-ara-AMP conjugate, the precipitated dpm ranged from aminimum of 41±5 to a maximum of 80±6 dpm.

This result showed that none of the treated mice produced antibodiesmeasurable with the employed method, the sensitivity of which was ofabout 0.5 μg/IgG/ml of serum.

e) Acute Toxicity of Compound 4

The Lac-poly-L-lysine-ara-AMP conjugate (No. 4), dissolved in 0.9% NaCl,was administered to female Swiss mice of 28-30 g intravenously (5animals) or subcutaneously (5 animals) with a single injection of 0.4ml/animal and in a 1.3 mg/g dose.

It did not show any toxicity evidence.

The injected dose was 50 times higher as compared with that administeredby intramuscular injection in the distribution experiments of theconjugate in the organs (FIG. 2).

The LD₅₀ for female Swiss mice of the poly-L-lysine utilized forproducing this conjugate, injected in the form of a hydrochloric acidsalt intravenously, resulted to be within 30 and 60 μg/g.

f) Solubility of the Compound 4

In patients with chronic hepatitis B the unconjugated ara-AMP isadministered at 2.5 or 5 mg/kg doses with two injections a day.

The Lac-poly-L-lysine-ara-AMP (No. 4) conjugate readily dissolves in aphysiological solution at the concentration of 400 mg/ml.

A 24 mg/kg, dose corresponding to 5 mg/kg of ara-AMP, can be thereforeadministered to a patient of 70 Kg weight in a volume lower than 5 ml.

B. Conjugate with Poly-L-Ornithine B.1 Preparation Compound 8

This compound was prepared using a poly-L-ornithine HBr (Sigma) with amolecular weight of 5,300-7,600. Poly-L-ornithine (25 mg) was labelledwith ³ H!formaldehyde (100 mCi/mmole)(NEN) according to Jentoft andDearbon (Methods Enzymol 1983, 91: 570-579). The reaction mixturecontained 98 μCi ³ H!formaldehyde/ml. ³ H!poly-L-ornithine was isolatedfrom the reaction mixture by gelfiltration on a Bio Gel P2 column elutedwith 0.5 M NH₄ HCO₃ and was subsequently lyophilized. Ara-AMPimidazolate was coupled to the labelled polymer using the procedurefollowed for compounds 3 and 4.

For subsequent lactosamination 10 mg ³ H!poly-L-ornithine-ara-AMP weredissolved in 1 ml 0.1 M borax/NaOH buffer, pH 10, together with 80 mgL-lactose and 50 mg NaBH₃ CN. The solution was incubated for 72 h at37°. The lactosaminated conjugate (Lac- ³ H!poly-L-ornithine-ara-AMP)was recovered by gel filtration on a Bio Gel P2 column eluted with 0.5 MNH₄ HCO₃ and was subsequently lyophilized. 1 mg compound (specificactivity 668 dpm/μg) contained 134 μg ara-AMP (spectrophotometricallydetermined) and 604 μg lactose (measured according to Dubois et al. AnalChem 1956; 28: 350-356). About 90% of polymer ε-amino groups weresubstituted: 17% with the drug, 73% with lactose.

B.2. Experimental Observations

a) Organ Distribution of Compound 8

FIG. 4 shows the organ distribution of radioactivity in mice afterintramuscular injection of Lac- ³ H!poly-L-ornithine-ara-AMP (36.5 μg/g,corresponding to 5 μg/g ara-AMP). Experimental procedure was asdescribed for the similar experiments with the at-poly-L-lysineconjugates. The levels of radioactivity in liver were higher than in theother organs. Radioactivity values in kidney were 3-4 times higher thanin spleen, intestine and brain.

II. HIGH MOLECULAR WEIGHT CONJUGATES

In patients with viral hepatitis the clearance of galactosyl terminatingmacromolecules is much slower than in mice, rats and normal humans,probably because of a slower penetration into hepatocytes (Marshall J S,Williams S, Jones P. Serum desialylated glycoproteins in patients withhepatobiliary dysfunctions. J Lab Clin Med 1978; 92: 30-37; TorraniCerenzia M R et al. J Hepatol 1994; 20: 307-309). As a consequence, inthese patients the renal elimination of the conjugates prepared with lowmolecular weight polyaminoacids is expected to be even greater than thatmeasured in mice.

To overcome this drawback we prepared conjugates using a high molecularweight poly-L-lysine. In these conjugates also most of the ε-aminogroups of poly-L-lysine were substituted by drug and galactosemolecules.

We observed that:

1) High molecular weight conjugates also specifically penetrate livercells after intramuscular administration.

2) The renal loss of these conjugates is very low and, consequently, thepercentage of injected dose entering hepatocytes is higher than thatmeasured after administration of low molecular weight conjugates.

3) High molecular weight conjugates are also devoid of acute toxicityand, after repeated intramuscular or intravenous administration, do notinduce antibodies.

4) Due to their high solubility (more than 150 mg/ml) and heavy drugload, a pharmacologically active dose could be administered in a smallvolume, well compatible with the intramuscular route.

II.1. Preparation

These conjugates were prepared using a poly-L-lysine HBr with amolecular weight of 30-70,000 and a polymerization degree of 145-335(Sigma). As in the preparation of low molecular weight poly-L-lysineconjugates drugs were coupled via the imidazolate of their phosphoricesters and lactose was linked by reductive amination in the presence ofNaBH₃ CN. However, the procedure was modified: the pH, temperature,length of reaction time as well as the imidazolate concentration wereall increased in the drug conjugation step. Moreover lactosamination wasperformed before drug coupling since at high pH high molecular weightpoly-L-lysine precipitated unless a part of ε-NH₂ groups weresubstituted with galactose residues.

Compound 9

Reductive lactosamination of ε-NH₂ groups was carried out by dissolving200 mg poly-L-lysine in 20 ml 0.4 M potassium phosphate buffer pH 7,together with 800 mg L-lactose and 500 mg NaBH₃ CN. After incubation at37° C. for 24 h the pH was raised to 8 with 5 M KOH and the solution wasleft at 37° C. for a further 6 h.

Lac-poly-L-lysine was diafiltered with 0.9% NaCl and concentrated to 100mg/ml. Lactose was measured by the phenol-sulphuric acid method ofDubois et al. (AnaI Chem 1956; 28: 350-356) using galactose as astandard; poly-lysine was determined by measuring the nitrogen accordingto Kjeldahl. 2 ml Lac-poly-L-lysine solution (=200 mg) were diluted with2 ml 1 M sodium carbonate buffer, pH 11. 800 mg ara-AMP imidazolate,synthesized according to Lohrmann and Orgel (Tetrahedron, 1978; 34:853-855), were dissolved and the pH was re-adjusted to 11 with 5 M NaOH.

After incubation at 50° C. for 96 h, the conjugate was diafiltered with0.9% NaCl. Chemical characterization of the complex was performed byassaying the coupled drug spectrophotometrically and by measuringlactose as described. The interference of ara-AMP in the colorimetricanalysis was subtracted. The poly-L-lysine content of conjugate wascalculated from the amount of lactose, knowing the weight ratiolactose/poly-L-lysine determined before drug coupling (see above). Thiswas possible because the bond between the sugar and lysine ε-NH₂ groupsdid not break down during drug conjugation, as we verifiedexperimentally. The conjugate was concentrated in saline (0.9% NaCl) to150 mg/ml and lyophilized after freezing to about -80° C. Theconcentration of conjugate was calculated without taking account ofcontra-ions.

Prior to use, Lac-poly-L-lysine-ara-AMP was dissolved with water at theconcentration of 150 mg/ml. It easily dissolved provided the freezingwas rapid. When necessary, the conjugate was diluted with 0.9% NaCl.

Compound 10

Prior to coupling with ara-AMP, Lac-poly-L-lysine was labelled with3H!formaldehyde (NEN) according to Jentoft and Dearbon (Methods Enzymol.1983; 91: 570-579). The reaction mixture contained 78 μCi ³H!formaldehyde/ml. After diafiltration with 0.9% NaCl Lac- ³H!poly-L-lysine was conjugated with ara-AMP as described above.

Compound 11

Ribavirin (RIBV)(1-β-D-ribofuranosyl- 1,2,4-triazole-3-carboxamide) wasfirst phosphorylated (RIBVMP) according to (Allen L B, Boswell K H,Khwaja T A, Meyer R B, Sidwell R W, Witkowski J T. Sinthesis andantiviral activity of some phosphates of the broad-spectrum antiviralnucleoside, 1-β-D-Ribofuranosyl-1,2,4-triazole-3-carboxamide (Ribavirin)J Med Chem 1978; 21: 742-746).

The pyridinium salt of the phosphorylated derivative was then convertedto the imidazolate (Lohrmann and Orgel, Tetrahedron 1978; 34: 853-855),which was coupled to Lac- ³ H!poly-L-lysine as described for theconjugate with ara-AMP. In this conjugate coupled RIBVMP was assayed bymeasuring the organic phosphate according to (Ames B N. Assay ofinorganic phosphate. Total phosphate and phosphatases. Methods Enzymol1966; 8: 115-118).

Due to the strong interference of RIBV with the colorimetric assay ofsugar, we could not measure lactose as described forLat-poly-L-lysine-ara-AMP and therefore we determined Lac- ³H!poly-L-lysine content of the conjugate by counting the radioactivity.

Compound 12

3'-azido-3- 2-¹⁴ C!deoxythymidine ( ¹⁴ C!AZT) (Moravek) wasphosphorylated according to Yoshikawa et al. (Tetrahedron Lett 1967; 50:5065-5068). The pyridinium salt of the phosphorylated derivative wasconverted to its imidazolate and subsequently coupled toLac-poly-L-lysine. Coupling was performed as described for compound 9,but in the reaction medium the ratio of the amount of drug imidazolateto that of the polymer was 2.7 instead of 4. The chemicalcharacterization of this conjugate was performed as described forcompound 9.

Compound 13

This conjugate was prepared as compound 9, but lactosamination ofpoly-L-lysine was interrupted after the first 24 h. Moreover, the ratioof the amount of ara-AMP imidazolate to that of the polymer was 4,6instead of 4. 30% of the ε-amino groups of the polymer was substitutedwith galactosyl residues and 64% with ara-AMP.

II.2. Experimental Observations

a) Average Molecular Weights of Compound 9

They were determined by permeation chromatography using HPLC equipment(Waters) with two Protein-Pak columns (125 and 300 SW) connected inseries. Compound 9 (80 μg) was dissolved in 20 ul mobile phase (125 mMNa₂ SO₄ +2 mM NaH₂ PO₄ H₂ O, to pH 6.0 with 0.1 N NaOH, filtered anddegassed) and chromatographed with the following conditions. Flow rate:0.9 ml/min; detection: UV at 260 nm, 0.1 absorbance units per full scale(AUFS). Columns were calibrated with aprotinin (Mr 6,500), RNAse (Mr13,700), HSA (Mr 69,000) and IgG (Mr 158,000). Weight average molecularweight and number average molecular weight were determined using the GPC745/745B Waters Software. They were found to be 140,339 and 72,419,respectively. The gel permeation chromatography of compound 9 is shownin FIG. 5.

b) Organ Distribution of Compounds

Experimental procedure was as described for the similar experiments withlow molecular weight conjugates. Results are shown in FIG. 6.

Conjugates of ara-AMP and RIBVMP were radioactive in the carrier whereasthe conjugate of AZTMP was labelled in the drug moiety.

After i.m. administration of the conjugates labelled in the carrier(FIG. 6, frames A, C) radioactivity was high in liver and low in spleen,intestine and kidney. The percentages of injected dpm recovered inkidneys were 10-20 times lower than those measured after i.m.administration of the complexes prepared with low molecular weightpoly-L-lysine. Since renal accumulation of proteins is a consequence oftheir glomerular filtration (Maack T H, Johnson V, Kau S T, FiguereidoJ, Sigulem D. Renal filtration, transport, and metabolism oflow-molecular weight proteins: A review. Kidney Int. 1979; 16: 251-270),the present result indicates that, as expected, only small amounts ofthe high molecular weight conjugates passed through the glomeruli atleast after i.m. administration (see below).

In mice i.m. injected with Lac-poly-L-lysine- ¹⁴ C!AZTMP the level ofradioactivity in liver was from 2.5 to 6 times higher than in kidneys,spleen and intestine (FIG. 6, frame D). The difference between theamount of radioactivity in liver and in other organs was less markedthan that in animals administered with the conjugates labelled in thecarrier (FIG. 6, Frames A,C). This result was probably due to a partialrelease of the drug (and/or its metabolites) from liver cells into thebloodstream after the intracellular cleavage of the drug-carrier bond. Asimilar release of the drug from hepatic cells in bloodstream wasobserved after administration of other drug/carrier hepatotropicconjugates (Fiume L, Busi C, Corzani S, Di Stefano G, Gervasi G B,Mattioli A. Organ distribution of a conjugate of adenine arabinosidemonophosphate with lactosaminated albumin in the rat.

J Hepatol 1994, in press; Fiume L, Mattioli A, Balboni P G, Tognon M,Barbanti-Brodano G, De Vries J and Wieland Th. Enhanced inhibition ofvirus DNA synthesis in hepatocytes by trifluorothymidine coupled toasialofetuin. FEBS Lett 1979; 103: 47-51).

When free ¹⁴ C!AZTMP was i.m. injected into mice the radioactivity wasequally distributed in liver, spleen and intestine with higher values inkidneys (FIG. 6, Frame E). The rate of accumulation and decline ofradioactivity after administration of free or coupled ¹⁴ C!AZTMP wasdifferent. After injection of the free drug, radioactivity accumulatedin tissues within the first 15 min and then rapidly declined; afterinjection of the conjugated drug radioactivity in liver increased up to4-5 h. At 1-2 h the amounts of radioactivity in liver were higher inanimals injected with conjugated ¹⁴ C!AZTMP at the dose of 2 μg/g thanin those administered with the free drug at 5 ug/g.

In mice injected intravenously with Lac- ³ H!poly-L-lysine-ara-AMP (FIG.6, Frame B) the conjugate rapidly accumulated in liver, in these animalsthe values of radioactivity in kidneys were higher than in those whichi.m. received the same conjugate (FIG. 6, Frame A). This can beexplained considering that:

(i) part of conjugate molecules had a molecular weight lower than thatof HSA (FIG. 5);

(ii) a direct relationship exists between plasma concentration andglomerular filtration of small proteins (Maack T H et al. Kidney Int1979; 16: 251-270);

(iii) the concentrations of conjugate in plasma which were constantlylow (less than 0.9 μg/ml) following the i.m. administration reached highvalues after the intravenous injection (104 μg/ml at 3 min).

c) Experiments of Tolerability and Immunogenicity

They were performed using compound 9. The conjugate administeredintravenously (i.v.) to 5 mice at the dose of 1.5 g/Kg did not cause anyrecognizable sign of suffering. 1.5 g of Lac-poly-L-lysine-ara-AMPcontained 480 mg of drug (see Table 2), a dose 300 times higher thanthat at which ara-AMP, when conjugated to L-HSA, inhibits virus growthin HBV-infected patients. In mice the LD₅₀ of poly-L-lysine used forpreparing the conjugate, given i.v. as salt of HCl, was between 15 and30 mg/Kg. To study whether Lac-poly-L-lysine-ara-AMP dissolved in salineat the concentration of 150 mg/ml can damage the tissues at the site ofadministration, a primary eye irritation experiment was performed in 6rabbits by placing 0.1 ml of the solution in the conjunctival sac. Noeye changes were observed in any animal.

Semithin sections of liver from mice and rats which receivedLac-poly-L-lysine-ara-AMP administered with different schedules (seeTable 3) were observed at light microscope. In none of the animals werechanges found in either parenchymal or sinusoidal liver cells.Accumulation into secondary lysosomes of non-completely digestedmolecules (disaccharides, peptides) which can not cross lysosomalmembrane results in a rapid swelling of these organelles which at lightmicroscope appear as cytoplasmic vacuoles. Such vacuoles were observedin hepatic cells of mice and rats 24 h after a single administration ofL-HSA-ara-AMP at doses 5-10 times higher than that active in HBVinfected patients (Fiume L, Betts C M, Busi C, Corzani S, Derenzini M,Di Stefano G, Mattioli A. The pathogenesis of vacuoles produced in ratand mouse liver cells by a conjugate of adenine arabinosidemonophosphate with lactosaminated albumin. J Hepatol 1992; 15: 314-322).The absence of vacuoles in liver cells of mice and rats afteradministration of high doses of Lac-poly-L-lysine-ara-AMP gave indirectevidence of a rapid digestion of this conjugate into products able tocross the lysosomal membrane.

To study the immunogenicity of Lac-poly-L-lysine-ara-AMP twenty fourmice received the conjugate for five days a week for four consecutiveweeks (single daily dose=200 μg/animal). Twelve mice wereintramuscularly injected while the others were administeredintravenously. A week after the last injection mice were bled andantibodies against the conjugate were measured as described for lowmolecular weight conjugates. None of the animals produced antibodies inamounts detectable by our assay (sensitivity about 0.5 μg IgG/ml serum).

From the previous experimental data it appears as most likely provedthat the antiviral nucleosides galatosylated poly-L-lysine conjugates,in which most of the ε-amino groups of the omopolimer are substituted bythe galactose residues and the drugs, administered by intramuscolarinjection, perform a hepatic targeting of drugs without producingantibodies.

They do not possess the acute toxicity of poly-L-lysine used for theirpreparation. In comparison with conjugates with the lactosaminatedalbumin, that need to be injected intravenously since they are otherwiseimmunogeus, the conjugates provided with the galactosylatedpoly-L-lysine are potentially able, in the infection by hepatiticviruses, to improve the patient compliance with a prolongedadministration of the antiviral agent.

As already mentioned, the foregoing referred to the conjugates whereinthe carrier was poly-L-lysine or poly-Lornithine; the experimentallyverified chemical-physical properties and biological behaviour makeacceptable the identical use as carriers of the other polyaminoacids.

These other conjugates fall therefore within the scope of the invention,as well as the employment of such polyamino acids as carriers forpreparing hepatotropic conjugates with antiviral compounds theadministration of which would be otherwise seriously compromised byunfavourable side phenomena induced by a high toxicity for organs otherthan liver.

                                      TABLE 2    __________________________________________________________________________    Characteristics of high molecular weight poly-L-lysine conjugates.                                        % ε-NH.sub.2 groups                      Lactose (μg)                               Drug (μg)                                        substituted by    Compound          Compound (mg)                               Compound (mg)                                        Lactose                                             Drug                                                 dpm/μg    __________________________________________________________________________    9 LAC-poly-L-lysine-ara-AMP                      385      330      48   43  0    10 LAC- .sup.3 H!poly-L-lysine-ara-AMP                      352      312      44   41  2910    11 LAC- .sup.3 H!poly-L-lysine-RIBVMP                      396      299      46   39  2504    12 LAC-poly-L-lysine- .sup.14 C!AZTMP                      371      219      45   26  466    13 LAC-poly-L-lysine-ara-AMP                      210      440      31   64  0    __________________________________________________________________________

                  TABLE 3    ______________________________________    Schedules of administration of Lac-poly(L-Lys)-ara-AMP to mice    and rats for the microscopic study of liver cells.            Daily        Route of Days of    Animal  dose (μg/g)                         injection                                  administration    ______________________________________    Mice    6            i.m.     20            30           i.v.     1            60           i.v.     1    Rats    6            i.m.     7            30           i.m.     7            60           i.v      1    ______________________________________

Animals were killed 24 h after the last injection. Liver samples werefixed and semithin sections were stained as described in (Fiume, L.,Betts, M. C., Busi, C., Corzani, S., Derenzini, M., Di Stefano, G.,Mattioli, A., J. Hepatol 1992; 15: 314-322).

We claim:
 1. A conjugate of a basic polyamino acid wherein most of the side chain amino groups of the basic polyamino acid bear either lactose or galactose residues and antiviral drug residues and the polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine.
 2. A conjugate according to claim 1, wherein the antiviral drug is selected from the group consisting of adenine-β-arabinoside 5'-monophosphate (ara-AMP), acyclovir, ribavirin and azido thymidine.
 3. A conjugate according to claim 1 which is Lac-poly-L-lysine-ara-AMP.
 4. A conjugate according to claim 1, which is Lac-poly-L-lysine-ACVMP.
 5. A conjugate according to claim 1, wherein at least 31% of the amino groups are substituted with lactose.
 6. A conjugate according to claim 1, which is Lac-poly-L-lysine-RIBVMP.
 7. A conjugate of a basic polyamino acid wherein most of the amino groups of said basic polyamino acid bear (a) galactose residues and (b) antiviral drug residues selected from the group consisting of ara-AMP, acyclovir, ribavirin and AZT.
 8. A conjugate according to claim 7, wherein at least 71% of the amino groups bear (a) galactose or lactose residues and (b) antiviral residues.
 9. An antiviral composition which contains as an active ingredient a pharmaceutically effective amount of a conjugate according to claim 1 in the presence of a pharmaceutical carrier.
 10. An antiviral composition according to claim 9, wherein the active ingredient is Lac-poly-L-lysine ACVMP.
 11. An antiviral composition according to claim 9 in a form suitable for intravenous, parenteral, subcutaneous or intramuscular administration.
 12. An antiviral composition according to claim 9, wherein the basic polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine.
 13. An antiviral composition according to claim 9, wherein said antiviral drug is selected from the group consisting of adenine-β-arabinoside 5'-monophosphate (ara-AMP), acyclovir, ribavirin and azidothymidine.
 14. An antiviral composition according to claim 9, wherein the active ingredient is Lac-poly-L-lysine-ara-AMP.
 15. A process for preparing a conjugate as defined in claim 13, which comprises the steps of:a) conjugation of the basic polyamino acid with an antiviral drug, and b) substitution on the amino groups with galactose residues by means of reductive lactosamination with cyanoborohydride.
 16. A process according to claim 15, wherein the drug conjugation is effected by means of the drug imidazolate and by performing the conjugation in a buffer medium in alkaline pH.
 17. A process according to claim 15, wherein the basic polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine.
 18. Process according to claim 15, wherein the antiviral drug is selected from the group consisting of adenine-β-arabinoside 5'-monophosphate (ara AMP), acyclovir, ribavirin and azidothymidine.
 19. A method for intramuscularly administering to a patient an antiviral effective amount of an antiviral composition comprising, as active ingredient, a conjugate of a basic polyamino acid wherein most of the amino groups of the basic polyamino acid bear either lactose or galactose residues and antiviral drug residues, wherein the polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine in a physiologically acceptable carrier, which comprises preparing a solution of said conjugate in a physiologically acceptable carrier and administering an antiviral effective amount thereof intramuscularly.
 20. A method for administering an antiviral composition according to claim 19 wherein the active ingredient is Lac-poly-L-lysine-RIBVMP.
 21. A method according to claim 19 wherein the physiological carrier is water.
 22. A method for subcutaneously administering to a patient an antiviral effective amount of an antiviral composition comprising, as active ingredient, a conjugate of a basic polyamino acid wherein most of the amino groups of the basic polyamino acid bear either lactose or galactose residues and antiviral drug residues, wherein the polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine in a physiologically acceptable carrier, which comprises preparing a solution of said conjugate in a physiologically acceptable carrier and administering an antiviral effective amount thereof subcutaneously.
 23. A method for administering an antiviral composition according to claim 22 wherein the active ingredient is Lac-poly-L-lysine-RIBVMP.
 24. A method according to claim 22 wherein the physiological carrier is water.
 25. A method for intravenously administering to a patient an antiviral effective amount of an antiviral composition comprising, as active ingredient, a conjugate of a basic polyamino acid wherein most of the amino groups of the basic polyamino acid bear either lactose or galactose residues and antiviral drug residues, wherein the polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine in a physiologically acceptable carrier, which comprises preparing a solution of said conjugate in a physiologically acceptable carrier and administering an antiviral effective amount thereof intravenously.
 26. A method for administering an antiviral composition according to claim 25 wherein the active ingredient is Lac-poly-L-lysine-RIBVMP.
 27. A method according to claim 25 wherein the physiological carrier is water.
 28. A method for parenterally administering to a patient an antiviral effective amount of an antiviral composition comprising, as active ingredient, a conjugate of a basic polyamino acid wherein most of the amino groups of the basic polyamino acid bear either lactose or galactose residues and antiviral drug residues, wherein the polyamino acid is selected from the group consisting of poly-L-lysine and poly-L-ornithine in a physiologically acceptable carrier, which comprises preparing a solution of said conjugate in a physiologically acceptable carrier and administering an antiviral effective amount thereof parenterally.
 29. A method for administering an antiviral composition according to claim 28 wherein the active ingredient is Lac-poly-L-lysine-RIBVMP.
 30. A method according to claim 28 wherein the physiological carrier is water. 